The long term objective of this project is an understanding, at a molecular level, of how the Na/K pump works. This virtually ubiquitous enzyme uses the energy from the hydrolysis of ATP to transport Na+ and K+ ions against their electrochemical gradient. By doing so, the Na/K pump maintains the K gradient that generates the transmembrane resting potential and the Na gradient which is used to produce electrical signals as well as to drive the uphill transport across the cell membrane of other substances. These ionic gradients are also responsible for cell volume regulation. Clinically, the Na/K pump is important because it is the receptor of digoxin, a widely prescribed cardiac steroid used to control some cardiac arrhythmias. In each pump cycle, three Na+ ions and two K+ ions are translocated through the protein. When ions are moved through the protein, electrical charge is translocated which can be measured and used to learn some properties of the movement of ions through the protein. To achieve the objective, voltage-induced steady and transient currents elicited by Na/K pumps will be measured in two preparations: the cloned rat brain pump expressed in a mammalian cell line and the endogenous pump from squid giant axons. For the recombinant rat brain pump, the possibility to manipulate the protein itself will be used to learn about the structure and function of the Na/K pump. Site-directed mutagenesis of selected residues to cysteine, and subsequent chemical modification, will be used to map the exposed surface of the protein. We will also exploit the interaction of palytoxin with the Na/K pump. Palytoxin is a marine toxin that is believed to transform the pump into a "channel", so that ions then move at rates 10,000 times faster than through the normal pumps. In the squid giant axon preparation, the access to internal and external solutions as well as the very fast voltage-clamp will be exploited to learn about the mechanism of ion translocation, particularly for Na+ ions. The three specific aims for this project are (i) to understand the ion-channel-like behavior by the Na/K pump, (ii) to determine the exposed surface of the pathways used by Na+ and K+ ions as they move through the protein, and (iii) to pursue the biophysical properties of the Na+ ion translocation mechanism through the Na/K pump.